Mutations in genes encoding synaptic proteins and impaired functional brain connectivity are emerging as common deficits associated with autism spectrum disorders (ASDs). However, how mutated synaptic proteins affect the properties of human neurons at the cellular and molecular levels to cause abnormal brain connections remains an important unanswered question. Addressing this question is essential for understanding the etiology and pathology of ASDs and developing novel and effective therapeutic strategies for patients. The PI previously demonstrated that SHANK3-deficient human cortical neurons derived from induced pluripotent stem cells (iPSCs), generated from 22q13 deletion syndrome patients with autism, have severely impaired intrinsic excitability and excitatory synaptic transmission. However, how these two phenotypes develop and affect neuronal connectivity in the brain remain unknown. The main goal of this project is to elucidate the cellular and molecular mechanisms responsible for development of synaptic and connectivity deficits in SHANK3-deficient human neurons. SHANK3 is a scaffolding protein expressed at excitatory synapses that have been frequently found to be mutated or deleted in individuals with autism and intellectual disability. The central hypothesis of this project is that synaptic deficits in SHANK3-deficient human neurons develop as a result of elevated electrical activity and activity-mediated weakening and elimination of excitatory synapses. This hypothesis is strongly supported by the preliminary data obtained in the PI's laboratory. The following Specific Aims are formulated to test this hypothesis: 1) Determine the role of elevated electrical activity in development of synaptic deficits in SHANK3-deficient human neurons; 2) Determine the role of ARC in development of synaptic deficits in SHANK3-deficient human neurons; and 3) Determine how loss of SHANK3 in human neurons impacts synaptic inputs onto these neurons in vivo. Under these aims, the properties of human cortical neurons generated from novel, precisely genetically-engineered stem cell lines will be investigated using electrophysiology, imaging, and biochemistry techniques in vitro and upon engraftment into the mouse brain. The proposed research is significant because it is expected to substantially advance understanding of the molecular, cellular, and circuitry mechanisms disrupted in autism and intellectual disability, and guide the development of novel therapeutic strategies for patients.